1. Field of the Invention
The present invention relates to a process for purifying an antibody composition having a desired property. Moreover, it relates to a medicament comprising the antibody composition obtained by the purifying method of the present invention as an active ingredient, and a method for treating various diseases using the antibody composition obtained by a substance having an affinity to the carbohydrate binding to the antibody.
2. Brief Description of the Background Art
Among glycopeptides, the glycopeptides whose carbohydrate structures coordinate toward surface of the proteins can be purified by means of a column to which a lectin binding to a carbohydrate is immobilized. A lectin has a property of specifically binding to a specific carbohydrate structure. Examples of the lectin include a wheat germ lectin, a lentil lectin and the like.
Upon the investigation of binding activity between a wheat germ lectin and a carbohydrate or glycopeptide, among N-glycans, a wheat germ lectin is suggested to have a high binding activity to a hybrid type carbohydrate or a carbohydrate or glycopeptide having sialic acid (Biochemistry, 16, 4426 (1977); The Journal of Biological Chemistry, 254, 4000 (1979)). Moreover, a wheat germ lectin is suggested to have stronger binding activity to glycopeptides having a carbohydrate structure having bisecting N-acetylglucosamine (Biochemistry, 20, 5894 (1981)).
A lentil lectin (hereinafter also referred to as “LCA”) is known to recognize monosaccharides, α-D-mannose and α-D-glucose (The Journal of Biological Chemistry, 268, 7668 (1993)). It is also known that LCA exhibits a strong binding activity to glycopeptides having a carbohydrate structure wherein L-fucose binds to an N-acetylglucosamine residue which is closest bound to the asparagine residue of N-glycan through α1,6-bond (Carbohydrate Research, 40, 111 (1975); Carbohydrate Research, 110, 283 (1982)).
However, these facts only indicate that lectins bind to carbohydrates or peptides containing a carbohydrate structure.
An antibody has a carbohydrate which binds to its Fc region (a region after the hinge region of the heavy chain of an antibody), and the carbohydrate is present in a form of being buried in the Fc region, i.e., in a form that the carbohydrate structure is structurally directed to inside of the antibody (Nature, 264, 415–420 (1976)).
Nose et al. have employed a column wherein LCA is immobilized to Sepharose carriers, but failed to separate mouse IgG2a. As a result, they considered that it is because the carbohydrate of usual mouse IgG2a produced by a hybridoma cell (12-6 cell) is buried in the Fc region. Also, they have cultured the hybridoma 12-6 cell after the addition of swainesonine which is an agent for inhibiting maturation of an N-glycan to produce IgG2a-class monoclonal antibody and passed the culture product through an LCA-immobilized Sepharose column, thereby the binding of the monoclonal antibody to the column being achieved. However, this is considered to be attributed to the exposure of the sugar chain out of Fc region of the antibody as a result of the conversion of the carbohydrate present in the Fc region of the mouse IgG2a from complex type to hybrid type by the effect of swainesonine (The Journal of immunology, 145, 910–914 (1990)).
As described above, the method for purifying an antibody by changing a carbohydrate structure artificially is known but the method for purifying an antibody in consideration of the carbohydrate structure without changing the carbohydrate structure is hitherto unknown.
By the way, the carbohydrate structure present in the Fc region of an antibody is involved in activities of antibody, specifically, antibody-dependent cell-mediated cytotoxic activity (hereinafter also referred to as “ADCC activity”), complement-dependent cytotoxic activity (hereinafter also referred to as “CDC activity”), in vivo stability, and the like.
It is known that the addition of galactose residue to a non-reducing end of a carbohydrate structure increases CDC activity of an antibody (Molecular Immunol., 32, 1311 (1995); WO98/58964), the increase of the content of bisecting N-acetylglucosamine-bound carbohydrate in Fc region of an antibody increases ADCC activity of the antibody (WO99/54342), and the increase of the content of sialic acid enhances in vivo stability (Nature Biotechnology, 17, 1116 (1999)). However, a process for purifying an antibody having a desired property such as effector activities including ADCC activity and CDC activity, or in vivo stability, while attention being paid to the carbohydrate structure relating to these activities is unknown hereto.
Also, in rheumatism, an autoimmune disease, the amount of galactose of IgG in patient's blood is known to decrease (Glycoconjugate Journal, 15, 929–934 (1998)). As a conventional diagnosis, a lectin blot method by a lectin is employed but the method requires complex operations including a step of modifying an antibody in a living body.